CAJ | 학술논문

目的观察大鼠阴茎海绵体肌源性干细胞(MDSCs)在血管内皮细胞生长因子(VEGF)和碱性成纤维细胞生长因子(bFGF)共同作用下向内皮细胞分化的潜能.方法采用Preplate差速贴壁法结合密度梯度离心法分离、纯化大鼠阴茎海绵体MDSCs,获得差速贴壁细胞(PP1-PP6),免疫荧光细胞化学法检测PP6细胞干细胞抗原-l(Sca-1)和结蛋白(Desmin)的表达,并传代培养.取PP6第3代细胞在VEGF和bFGF共同诱导21 d后,免疫荧光细胞化学法鉴定内皮细胞标志物[CD31、胎肝激酶-l(Flk-1)]的表达；分别在诱导0、5、10、15、21 d,通过逆转录-聚合酶链反应(RT-PCR)检测干细胞标志物(Oct-4)和内皮细胞标志物[CD31、内皮型一氧化氮合酶(eNOS)、Flk-1] mRNA的表达量；通过Dil-Ac-LDL摄取实验检测细胞吞噬低密度脂蛋白的能力；划痕24h后观察划痕空白处的距离,评估细胞的迁移能力.结果 PP6细胞表达肌源性干细胞标志物Sca-1和Desmin；PP6诱导21 d后表达内皮细胞标志物(CD31和Flk-1),诱导21 d后与诱导0d比较,干细胞标志物Oct-4的mRNA表达量下降(下降倍数为0.445±0.025),而内皮细胞标志物mRNA表达量升高(CD31、eNOS、Flk-1升高倍数分别为2.541±0.146、2.364±0.175、2.538±0.100),差异均有统计学意义(P＜0.05).Dil-Ac-LDL摄取实验提示诱导后的细胞具有吞噬低密度脂蛋白的能力；划痕实验观察到诱导组空白处距离为282.01±3.84,未诱导组空白处距离为450.35±1.86,差异有统计学意义(P＜0.05).结论大鼠阴茎海绵体MDSCs在VEGF和bFGF的共同诱导下可分化为内皮细胞,且具有成熟内皮细胞的功能.
목적 관찰대서음경해면체기원성간세포(MDSCs)재혈관내피세포생장인자(VEGF)화감성성섬유세포생장인자(bFGF)공동작용하향내피세포분화적잠능.방법 채용Preplate차속첩벽법결합밀도제도리심법분리、순화대서음경해면체MDSCs,획득차속첩벽세포(PP1-PP6),면역형광세포화학법검측PP6세포간세포항원-l(Sca-1)화결단백(Desmin)적표체,병전대배양.취PP6제3대세포재VEGF화bFGF공동유도21 d후,면역형광세포화학법감정내피세포표지물[CD31、태간격매-l(Flk-1)]적표체；분별재유도0、5、10、15、21 d,통과역전록-취합매련반응(RT-PCR)검측간세포표지물(Oct-4)화내피세포표지물[CD31、내피형일양화담합매(eNOS)、Flk-1] mRNA적표체량；통과Dil-Ac-LDL섭취실험검측세포탄서저밀도지단백적능력；화흔24h후관찰화흔공백처적거리,평고세포적천이능력.결과 PP6세포표체기원성간세포표지물Sca-1화Desmin；PP6유도21 d후표체내피세포표지물(CD31화Flk-1),유도21 d후여유도0d비교,간세포표지물Oct-4적mRNA표체량하강(하강배수위0.445±0.025),이내피세포표지물mRNA표체량승고(CD31、eNOS、Flk-1승고배수분별위2.541±0.146、2.364±0.175、2.538±0.100),차이균유통계학의의(P＜0.05).Dil-Ac-LDL섭취실험제시유도후적세포구유탄서저밀도지단백적능력；화흔실험관찰도유도조공백처거리위282.01±3.84,미유도조공백처거리위450.35±1.86,차이유통계학의의(P＜0.05).결론 대서음경해면체MDSCs재VEGF화bFGF적공동유도하가분화위내피세포,차구유성숙내피세포적공능.
Objective To observe the potential of the rat penile corpus cavernosum muscle-derived stem cells (MDSCs) differentiation into endothelial cells (ECs) in the presence of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in vitro.Methods MDSCs were purified through density gradient centrifugation and differential attachment.PP1-PP6 cells were obtained,and the expression of stem cell marker Sea-1 and myoblast marker Desmim in PP6 cells was detected by using immunofluorescence.The PP6 cells at third passage were induced to differentiate into endothelial cells within 21 days after VEGF and bFGF stimulation.The differentiating MDSCs were assessed by using immunofluorescence of ECs markers [CD31,fetal liver kinase-1 (Flk-1)],reverse transcriptase-polymerase chain reaction (RT-PCR) of ECs markers [CD31,endothelial nitric oxide synthase (eNOS),Flk-1] and stem cell markers (Oct-4) at 0,5,10,15 and 21 days.Dil-Ac-LDL uptake test was used to test the ability of cells taking up acetylated LDL,and scratch tests was used to observe the scratch space distance after 24 h.Results We observed that PP6 cells expressed MDSCs markers (Desmin,Sca-1) and the differentiating MDSCs expressed ECs markers (CD31,Flk-1) within 21 days after VEGF and bFGF stimulation.As compared with 0 day,the expression of Oct-4 was declined by 0.445 ± 0.025 times,and that of ECs-specific marker mRNA was significantly increased in the differentiated MDSCs at 21 days by 2.541 ± 0.146,2.364 ± 0.175 and 2.538 ± 0.100 times for C D31,eNOS and Flk-1,respectively (P ＜ 0.05 for all).An increased uptake of Dil-Ac-LDL in MDSCs-derived ECs was observed at 21 days compared to undifferentiated MDSCs,and the migration ability of MDSCs-derived ECs was significantly increased.The scratch space distance of differentiated MDSCs was 282.01 ± 3.84 and that of undifferentiated MDSCs was 450.35 ± 1.86 with the difference between statistically significant (P ＜ 0.05).Conclusion The rat penile corpus cavernosum MDSCs can differentiate into ECs in the presence of VEGF and bFGF in vitro and differentiated MDSCs have the typical functions for mature ECs.